The present invention relates to compounds and compositions that are potent and selective inhibitors of the aromatase enzyme system, and in particular, to aromatase inhibitors extracted from Broussonetia papyrifera. The present invention also relates to cancer chemopreventive compositions and methods. More particularly, the present invention relates to cancer chemoprevention in mammals, including humans. The present invention further relates to methods of treating conditions and diseases wherein inhibition of aromatase provides a benefit, such as breast cancer, prostate cancer, and other hormone-dependent cancers.
Chemoprevention, i.e., the prevention of cancer by administration of chemical agents that reduce the risk of carcinogenesis is one of the most direct ways to reduce cancer-related morbidity and mortality. See, M. B. Sporn, Fed. Proc., 38, p. 2528 (1979). However, chemoprevention requires the identification of carcinogens and chemopreventatives, even though interactions between the factors that modulate cancer risk are complex. Whereas extensive efforts have been made to identify carcinogens and mutagens, the identification of chemopreventative agents has received less attention.
Cancer chemopreventive agents include nonsteroidal antiinflammatory drugs (NSAIDs), such as indomethacin, aspirin, piroxicam, and sulindac, all of which inhibit cyclooxygenase. There is a need in the art, however, for the identification of additional specific compounds that have a cancer chemopreventative effect on mammals. To this end, investigators have searched for new cancer chemopreventative agents by evaluating hundreds of plant extracts for a potentially active compounds. Such cancer chemopreventative compounds then can be used in drug compositions to reduce the risk of, or to treat, a cancer, such as breast or prostate cancer, and other hormone-dependent cancers.
Breast cancer is a major cause of morbidity and mortality in women throughout the world. Epidemiological and experimental evidence strongly support a role for estrogens in the development and growth of breast cancer. A role for estrogens in prostate neoplasia has also been postulated. Therefore, one chemotherapeutic or chemopreventive strategy for breast cancer and prostate cancer control is to decrease estrogen production.
An alternative approach to the endocrine treatment of estrogen-dependent breast cancer is the reduction of the supply of estrogens to the tumor. In the treatment of hormone-dependent breast cancer, estrogen receptors can be blocked with antagonists, such as tamoxifen. Gonadotrophin can be inhibited by a continuous administration of gonadatrophin-releasing hormone (GnRH) or one of its analogues. P. M. Conn et al., N. Engl. J. Med., 324, pp. 93-103 (1991). Estrogens are biosynthesized from androgens by a microsomal cytochrome P450 enzyme complex system termed aromatase. P. K. Siiteri, Cancer Res., 42, (Suppl. 8), pp. 3269s-3273s (1982).
Accordingly, the inhibition of aromatase, an enzyme that catalyzes the final, rate-limiting step in estrogen biosynthesis, is an effective approach in the treatment of breast and prostate cancers. The use of aromatase inhibitors is of clinical interest for cancer therapy, and selective and potent aromatase inhibitors have been developed.
Recent studies on the fundamental biologic properties of the very complex aromatase enzyme system yielded information concerning genetic control, differential tissue expression, and modulation by a variety of growth factors and cytokines present within the tumor environment. H. A. Harvey, Semin. Oncol., 23 (Suppl. 9), pp. 33-38 (1996). Using monoclonal or polyclonal antibodies against aromatase, it has been demonstrated that a significant amount of aromatase accumulates in breast tumors and the stroma surrounding breast tumors. R. J. Santen et al., Endocr. Rev., 11, 221-265 (1994). This finding may be of relevance in the initiation of breast carcinoma, and in the development of compounds of sufficient potency to effectively lower plasma levels of estrogen by inhibiting estrogen synthesis in situ. Because estrogen production is the last step in the biosynthetic sequence of steroid production, selective inhibition of aromatase would not interfere with the production of other steroids, such as adrenal corticoids. For these reasons, aromatase is a particularly attractive enzyme target for selective inhibition in the treatment of cancers.
Several classes of aromatase inhibitors, such as substrate androstenedione derivatives, the nonsteroidal aminoglutethimide and its analogues, imidazoles, and triazoles, have been developed over the past twenty years as potential therapeutic agents. G. J. Kelloff et al., Cancer Epidemiol. Biomark. Prev., 7, pp. 65-78 (1998). Aminoglutethimide (AG) was the first aromatase inhibitor used clinically. AG effectively reduces N-methyl-N-nitrosourea (MNU)-induced tumor incidence in Sprague-Dawley rats when administered at 400 mg/kg diet. However, the nonselectivity of this compound to aromatase, its structural similarity to phenobarbital and the associated CNS effects, and lack of potency compared to the triazole aromatase inhibitors has led to its decreasing use. Rogletimide, an AG analogue, is a more specific, but less potent aromatase inhibitor than AG, and also is effective in reducing the testosterone-induced increase in tumor size.
Several other triazole, nonsteroidal aromatase inhibitors are effective aromatase inhibitors. For example, vorozole is one of the most potent and specific aromatase inhibitors, in vitro and in vivo. At p.o. (per os, or oral) doses of 5 mg/kg, vorozole decreases the % tumor incidence from 100 to 10, and the tumor multiplicity from 5 to 0.1 tumors/animal, in Sprague-Dawley rats. However, one major disadvantage of vorozole is that it has a weak androgenic activity and, as a result, the treated animals appeared bulky and heavily muscled. Among the steroidal inhibitors, exemustane (administered subcutaneously) was effective in causing tumor regression and preventing the formation of new tumors. Exemustane, however, has androgenic effects similar to vorozole.
The present invention is directed to potent and selective aromatase inhibitors that do not exhibit the disadvantages and drawbacks associated with prior aromatase inhibitors.
In a programmed research effort to discover novel, natural product-based cancer chemopreventive agents, i.e., agents capable of preventing, inhibiting, or reversing the process of carcinogenesis, about four thousand plant samples from around the world were extracted with an organic solvent, and the dried extracts so produced were tested in an array of bioassays reflective of modulating carcinogenesis at the stage of initiation, promotion, or progression. As a result of this research, an ethyl acetate-soluble extract of a domestic plant called Broussonetia papyrifera was found to exhibit a potent aromatase inhibitory activity.
Broussonetia papyrifera is a deciduous tree that is naturalized in the United States, parts of which have been used for treatment of impotency and ophthalmic disorders in the People""s Republic of China. In a search for edible aromatase inhibitors, an extract of B. papyrifera was found to have significant activity (0.4 xcexcg/ml). Extracts of B. papyrifera have shown antifungal, antihepatotoxic, antioxidant, and aldose reductase inhibitory activities. Also, several flavonoid constituents of this plant have been shown to inhibit lipid peroxidation and to exhibit antiplatelet effects.
Bioassay-guided fractionation of a Broussonetia papyrifera extract using an in vitro aromatase inhibition assay led to the isolation of both novel and known active compounds. The compounds included coumarins, benzofurans, biphenylpropanes, and various types of flavonoids.
Flavonoids are natural components which are present in many plants known to be constituents of animal and human diet. These compounds exhibit a variety of biological properties, such as antiviral, antiinflamatory, antimutagenic, and anticarcinogenic activities. J. D. LeBail et al., Cancer Lett., 133, pp. 101-106 (1998). Studies have shown that some flavones, isoflavones, and flavanones alter hormone production and inhibit aromatase activity in human and ovarian microsomes. In addition, due to their structural similarity to estrogens, some flavonoids bind to the estrogen receptor and possess mixed agonist/antagonist properties. B. M. Collins et al., Steroids, 62, pp. 365-372 (1997).
Based on this observation, purification of the constituents of the plant extract responsible for the biological activity was traced using an in vitro aromatase inhibition assay, and identified using spectroscopic techniques to a first flavonoid of novel structure, (2S)-2xe2x80x2,4xe2x80x2-dihydroxy-2xe2x80x3-(1-hydroxy-1-methylethyl)-dihydrofuro[2,3-h]flavanone (11), and a second flavonoid of previously known structure, isolicoflavonol (i.e., 3,5,7,4xe2x80x2-tetrahydroxy-3xe2x80x2-prenylflavone) (12). 
Compounds 11 and 12 (at about 0.1 xcexcM) exhibited an approximately 60-fold greater potency for aromatase inhibitors compared to aminoglutethimide (AG), the positive control used for this assay. Initially, some of the compounds isolated from B. papyrifera were tested for binding to the estrogen receptor-xcex1 or -xcex2. J. A. Gustafsson, J. Endocrinol., 163, pp. 379-383 (1999). Interestingly, none of the aromatase active compounds showed significant binding to either of the receptors. Also, the effectiveness of some of the flavonoids against the inhibition of quinone reductase, a phase II enzyme involved in detoxification mechanisms, was evaluated. L. C. Chang et al., J. Nat. Prod., 60, pp. 869-873 (1997); and P. Talalay, Proc. Natl. Acad. Sci. USA, 85, pp. 8261-8265 (1998). No significant inhibition of aromatase was observed by any of the agents tested. Compound 11 also was effective in inhibiting (50%) the formation of alveolar lesions in a mouse mammary organ culture model when tested at 100 ng/ml. R. G. Mehta et al., Anticancer Res., 14, pp. 1209-1213 (1994).
The present invention, therefore, is directed to the chemotherapy or chemoprevention of estrogen-dependent breast and prostate cancers. While even more potent aromatase inhibitors have been developed, the compounds isolated from B. papyrifera demonstrated a very potent inhibition of aromatase, and are isolated from a common, natural, and renewable source. Also, the compounds from B. papyrifera are based on nonsteroidal moieties, unlike any current class of inhibitors, and are natural products that have reduced toxicity and, therefore, a wider acceptance in the general population.
The present invention, therefore, is directed to cancer chemopreventative agents, compositions containing the agents, and methods of using the chemopreventative agents to prevent and/or treat a cancer, like breast cancer or prostate cancer. In particular, the present invention is directed to compositions containing one or more compounds found in an extract from Broussonetia papyrifera and that are capable of inhibiting the aromatase enzyme system, and use of the compositions in methods of cancer chemoprevention.
An important aspect of the present invention, therefore, is to provide a method and composition for preventing or treating a cancer using one or more compounds found in an extract from Broussonetia papyrifera and capable of inhibiting the aromatase enzyme system.
Another aspect of the present invention is to overcome the problem of high mammalian toxicity associated with present cancer chemopreventative agents by using a natural product-derived compound.
Still another aspect of the present invention is to overcome the problem of insufficient availability associated with synthetic anticancer agents by utilizing readily available, and naturally occurring, chemopreventative agent.
Another important aspect of the present invention is to provide a drug composition containing one or more compounds found in an extract from Broussonetia papyrifera and capable of inhibiting the aromatase enzyme system, and that can be administered to chemoprevent cancers.
These and other aspects of the present invention will become apparent from the following detailed description of the preferred embodiments of the invention.